The present invention relates broadly to surface plasmon resonance detection of analytes. The invention further relates to methods employing such apparatus.
The use of Surface Plasmon Resonance (SPR) for the detection of small soluble analytas from solution is well known (see e.g. xe2x80x9cAdvance in Biosensorsxe2x80x94A Research Annual Vol 1. 1991xe2x80x9d Ed. A P F Turner, Pub. Jai Press Ltd, London).
CB 2 271 886 (Secretary of State for Defence), to which reference should be made, discloses SPR apparatus comprising a light source for generating polarised light, a sensor, the outside of which is metal coated and may be contacted with a sample solution, and means for detecting the light which is internally reflected from the inner sensor surface.
In the absence of bound analyte, light is totally internally reflected at an incident angle characteristic of the refractive index (RI) of the sensor and of the sample solution. At a particular incident angel (the SPR angle), interaction of the metal with the evanescent wave set up by internal refection of the polarised light causes a drop in intensity of the reflected light. This drop can be observed using the light detector.
The binding of analyte to the sensor surface, within the evanescent wave zone, alters the RI of the area above the sensor and this perturbs the SPR angle. This perturbation can be observed using the light sensor and related to the surface concentration of analyte.
SPR detection in the literature has generally been limited to use with soluble molecular size analytes e.g. biomolecules such as proteins and nucleic acids which are specifically bound within the evanescent zone using appropriate liganda.
According to a first aspect of the present invention, surface plasmon resonance apparatus for detecting a soluble and/or a particular analyte is characterised in that it comprises:
(a) a sensor providing a metallised sensor surface capable of binding the analyte:
(b) a light source excitation beam for direction at the sensor surface:
(c) at least one detector capable of detecting light from the light beam which is internally reflected from the sensor surface, and
(d) beam modifying means whereby the excitation beam is influenced in a controlled manner whereby the level of light emitted from the sensor surface is substantially enhanced.
The beam modifying means may be operable so as to displace the excitation beam over an angular range, relative to the sensor surface.
Alternatively, the beam modifying means may be operable so as to adjust the wavelength of the excitation beam.
According to a second aspect of the present invention, a method of detecting an analyte in a sample comprises exposing said sample to a metallised sensor surface and interpreting the light signals emitted from the sensor surface.
Possible analytes may include those particular or insoluble analytes containing or consisting of biomolecules, for instance bacteria or other cells, spores, virions etc., or biomolecules themselves such as proteins and polynucleotides.
Possible targets include cryptosporidium, E. coli, salmonella etc.
The excitation displacement means may comprise a beam-reflecting mirror and means for vibrating the mirror. The exitation beam may comprise a linear-beam, a fan-shaped beam or a wedge-shaped beam.
The excitation angle of displacement may be automatically adjustable according to changes detected in the SPR signal, thereby keeping the apparatus xe2x80x9con-resonancexe2x80x9d. Further adjustment may be required so as to take into account different areas of a matrixed sensor surface.
It will be advantageous to provide means for monitoring changes in resonance and light scattering emission so as to maintain a preferred angle of incidence.
Automatic scanning is preferred, whereby at any given position on the sensor surface the required condition for resonance and therefore substantially maximum scatter emission will be achieved.
Maximum surface emission conditions may be obtainable by use of appropriate controlled software, whereby, for example, each defined area of a matrixed surface during each single angle scan would take place in a time period less than 1.0 second.
A third aspect according to the invention comprises a microscope incorporating detection apparatus as hereinbefore described.